P R O T E I N
S T U D I E S
Protein Expression,
Folding and Purification
Summary and Specifications
pCold TF DNA
Thiophilic Resins, Purification of Antibodies
Glycoprotein Enrichment and Detection Kits, Enrichment and Detection of Glycoproteins
Phosphoprotein Enrichment Kit
Separation of Phosphorylated and Unphosphorylated Proteins
Phosphopeptide Enrichment Kit
Enrichment of Phosphopeptides
ProteoGuard-Protease Inhibitor Cocktail
TALON, High Purity Purification of His-Tagged Proteins
His60 , High Yield Purification of His-Tagged Proteins
DYKDDDDK Beads (FLAG Epitope)
Purification and Immunoprecipitation of DYKDDDDK (FLAG Epitope) Tagged Proteins
c-Myc Agarose Beads
Purification and Immunoprecipitation of Myc-Tagged Proteins
HA-Tag Antibody
Immunoprecipitation and Detection of HA-Tagged Proteins
GST Purification Resin Purification of GST-Tagged Proteins
Other Purification Products
Purification of Tagged Proteins
Expression in Hard-to-transfect Dividing Cells
Retroviral Expression Systems
Adenoviral Expression Systems Transient Expression
in many Dividing and Nondividing Mammalian Cell Types
Expression in many Dividing and Nondividing Cell Types
Lentiviral Expression Systems
Inducible Expression of Proteins
Inducible Vectors
Expression of Two Proteins at Similar Level
Bidirectional Vectors
Expression of Two Proteins from One RNA
Maximize Soluble Active
Protein
S T U D I E S
Protein Purification Products
Expression and Purification of
His-tagged Proteins
Bicistronic Vectors
Expression of HA-tagged Proteins
pET Express & Purify Kits
Ha Vectors
cMyc-tagged Proteins
Myc Vectors Expression
Expression of DYKDDDDK-tagged Proteins
Chaperone Plasmid Set
Reforming Disulfide Bonds
DYKDDDDK (FLAG Epitope)Vectors
Corystein™ Reagent
Optimize Refolding
of Inclusion Bodies
Strong Promoter for Increased Expression in many cell types
Refolding CA Kit
Increase Level of Active
Protein
Refolding Insoluble
Protein
Chaperonin GroE
Chaperone Assisted
Folding
Folding
EF1a Vectors
All-purpose Gene Expression Vectors
CMV Vectors
Mammalian Expression
MazF Enzyme
Efficient Protein Production and
Posttranslational Modifications
Baculovirus Expression System
High yield (posttranslational
modifications)
Secretory Protein Production
Bacillus Subtilis Expression
System
High Efficiency Cell-Free
Protein Production
Human Cell Free
Expression System
Cell Free protein
Expression
mRNA Interferase™-Plasmid
Single Protein Production System
SPP System™ Kits
Express Soluble Fusion Proteins
pCold Pro S2
Increase Expression using Trigger
Factor
High Efficiency Protein Production
Brevibacillus Expression
System II
pCold Vectors
Increase Recombinant Protein Yield
High Yield Active Protein
Increase Yield and Purity in E.coli
Expression
Protein Expression and Folding Products
P R O T E I N
O V E R V I E W
E X P R E S S I O N
S Y S T E M S
Bacterial Expression & Purification—pET Express &
Purify Kits
• Powerful—Higher inducible protein expression levels & tighter control of your target gene
pET Express & Purify Kits—a Complete
Expression & Purification System
• Fast—E. coli BL21(DE3)-based system
The pET Express & Purify kits supply a choice of IMAC resins
and buffers to purify expressed his-tagged proteins. You can
choose His60 Ni nickel-based resin for high binding capacity
or HisTALON cobalt-based resin for high purity. The kits are
supplied with prepacked gravity columns filled with either resin
and provide all of the buffers necessary to perform the protein
extraction and purification.
• Versatile—Choose between N- & C-terminal 6xHN-tagged vectors
• Convenient—Choice of fast, simple In-Fusion PCR cloning or traditional T4 DNA ligase cloning
• Complete—Purify your expressed protein with His60 Nickel resin or TALON® Cobalt resin
The pET expression system is the most commonly used
bacterial system for the over-expression of genes. We offer a
complete system with a choice of N- or C-terminal 6xHNtagged vectors together with IMAC-based purification. Our
pET system utilizes two levels of regulation to provide the
highest level of protein expression and the tightest control over
basal expression—achieved via the presence of lac operator
sites in two different promoters.
Cobalt-Based Resin—HisTALON
• The first level of regulation is provided by the pET6xHN
series of vectors. The gene of interest is cloned downstream
of a strong T7 lac hybrid promoter, which combines the
T7 promoter with the lac operator. T7 RNA polymerase
is extremely selective in binding to this hybrid promoter,
thereby utilizing most of the cell’s resources to express this
gene.
Nickel-Based Resin—His60 Ni Superflow Resin
• The second level of regulation occurs in the host cell. The
T7 RNA polymerase gene is integrated into the host genome
under the control of the lac UV5 promoter, which also
contains a lac operator. This enables expression of T7 RNA
polymerase to be controlled by the lacI genes present in both
the host genome and the pET6xHN vectors, which encode
lac repressor.
• No copurification of unwanted host proteins
• No SlyD contamination (histidine-rich protein present
in E. coli)
• Lowest metal ion leakage
• Available in various formats as bulk resin, cartridges, and
gravity columns
• 60 mg/ml binding capacity
• Up to 95% purity
• Low metal ion leakage
• Available in various formats as bulk resin, cartridges, and
gravity columns
In the uninduced state lac repressor inhibits expression of both
T7 RNA polymerase and the gene of interest. When IPTG is
added during induction, it binds to lac repressor, which then
dissociates from the lac operators, removing this inhibition.
This allows expression of T7 RNA polymerase, which in
turn binds to the newly derepressed T7 lac hybrid promoter
and transcribes the gene of interest. The RNA transcript is
then translated, leading to a very high level of target protein
expression within the host cell.
The pET Express & Purify System Vectors—
pET6xHN
Clontech’s pET Express and Purify kits contain pET vectors
(the pET6XHN series of vectors) which encode N- or
C-terminal 6xHN fusion tags. These are available in a choice
of cloning formats (easy In-Fusion cloning or traditional
restriction enzyme cloning) for maximum flexibility tailored to
your expression needs.
The pET Express & Purify Kit protocol.
Product Information
Brand
Product
Size
Cat.No
Clontech
pET Express & Purify Kit—His60
20 Purifications
631431
Clontech
pET Express & Purify Kit—His60 (In-Fusion Ready)
20 Purifications
631428
Clontech
pET Express & Purify Kit—HisTALON
20 Purifications
631430
Clontech
pET Express & Purify Kit—HisTALON (In-Fusion Ready)
20 Purifications
631429
4
E X P R E S S I O N
S Y S T E M S
B. subtilis Secretory Protein Expression System
Features
• Includes pBE-S DNA, an E. coli/B. subtilis shuttle vector with B. subtilis-derived subtilisin (aprE) promoter, secretory signal peptide (aprE SP), Multiple Cloning Site, and 3’ (C-terminal) His-tag sequence
• Supplied with SP DNA Mixture, a library of DNA sequences encoding 173 unique secretory signal peptides that can be inserted upstream of your target gene
• Fully compatible with In-Fusion cloning kits and systems to allow rapid and easy construct generation
Optimization of secretion, however, can be necessary to
achieve highest yields. To address this, the B. subtilis Secretory
Protein Expression System from Takara Bio allows rapid
development of a library of B. subtilis clones, each bearing a
pBE-S construct in which the ORF for your protein of interest
is fused with sequences for 173 unique signal peptides. Perform
a downstream assay to identify and select clones which secrete
the highest amount of functional protein into the culture
media, and you can quickly identify the signal peptide that
results in efficient expression of your desired secreted protein.
• Includes B. subtilis strain RIK1285
Applications
pUB
His-Tag
(5,938 bp)
Kanr
ColE1
Ampr
• Expression of proteins with complex structure, such as
proteins with disulfide (S-S) bonds
Vector map for pBE-S DNA, a B. subtilis/E. coli shuttle
vector used with the B. subtilis Secretory Protein
Expression System.
A405
A405
A405
• Generation of target protein in a host that is considered to
be Generally Regarded As Safe (GRAS) by the U.S. Food
and Drug Administration
Bacillus subtilis has become an increasingly popular host
for recombinant protein expression. With its ability to
secrete protein directly into culture media, amenability to
medium- and large-scale fermentation, lack of codon bias,
and designation by the U.S. Food and Drug Administration
as an organism that is Generally Regarded As Safe (GRAS),
it’s no wonder that the majority of industrially-produced
enzymes are expressed in Bacillus species such as B. subtilis.
multi cloning site (MCS)
pBE-S DNA
• Protein expression in a host amenable to medium- and largescale fermentation in addition to small-scale culturing
Description
52 I
SP
• Expression of soluble, recombinant protein secreted directly
into the culture media
• Useful for producing easily purified recombinant protein –
with proper in-frame cloning, a C-terminal His-tag can aid
purification from culture media
173 different types of SP DNA
are inserted into this region
in place of the
SP
I
promoter
3
2.5
2
1.5
1
0.5
0
1
3
2.5
2
1.5
1
0.5
0
161
3
2.5
2
1.5
1
0.5
0
321
11
21
31
41
51
61
71
81
91
101
111
121
131
141
151
◄
171
181
191
201
211
221
231
241
251
261
271
281
291
301
311
◄
331
341
351
361
371
381
391
401
411
421
431
441
451
461
◄
䠝㻮
Results of measuring β-glycosidase activity of 470 clones of an expression
library with different signal peptide sequences.Clones showing activity
levels of varying strengths were observed. The arrowheads indicate the
expression level observed with the aprE signal peptide.
Related In-Fusion Cloning Products
For rapid and easy cloning, use the In-Fusion HD Cloning System: see page TBD
Product Information
Brand
Product
Size
Cat.No
Takara
B. subtilis Secretory Protein Expression System
10 rxns
3380
Flowchart of the experimental procedure for the
B. subtilis Secretory Protein Expression System.
5
E X P R E S S I O N
S Y S T E M S
Brevibacillus Expression System II
Features
• Efficient production of secreted or intracellular target proteins
• Produces negligible amounts of extracellular protease – products remain intact in culture medium
• Unlike E. coli, produces no endotoxins
• Proteins are produced in active form
• Easy to culture, handle, and sterilize
Description
Brevibacillus choshinensis is a gram-positive bacterium with
exceptional capacity for heterologous protein expression.
The Brevibacillus Expression System II enables highly efficient
production of target protein in secreted form. This system
allows high yield of active proteins and is well-suited for
expression of eukaryotic proteins. The Brevibacillus system is
nearly free of proteases, which facilitates production of intact
protein products.
Examples of successfully expressed proteins can be seen in
Table 1. This includes expression of enzymes, antigens, and
cytokines. Each protein was produced at a very high level of
expression and confirmed to have native biological activity.
In addition, proteins from taxonomically distant organisms
were successfully produced, such as eubacteria, archaebacteria,
eukaryotes, and viruses.
The Brevibacillus system facilitates disulfide bond formation
(commonly required in proteins of eukaryotic origin).
In addition, B. choshinensis serves as an excellent host for
intracellular protein production, frequently producing
intracellular proteins in soluble form in the cytoplasm without
forming inclusion bodies. The Brevibacillus system often works
better than E. coli for expression of particular targets.
Utilizing His-tag containing vectors (pNC-HisE, pNC-HisF,
pNC-HisT, pNI-His) allows effective purification of the
expressed target protein. Tags can be removed by protease
treatment following purification.
Proteins
Enzymes
α-amylase
α-amylase
Sphingomyelinase
Sphingomyelinase
Xylanase
Xylanase
CGTase
CGTase
Chitosanase
Chitosanase
Hyper
Hyperthermo-stable
thermo-stableprotease
protease
Hyper
Hyperthermo-stable
thermo-stablenuclease
nuclease
PDI
PDI
Antigens
Surface
Surfaceantigen
antigen
Surface
Surfaceantigen
antigen
Cytokines
EGF
EGF
IL-2
IL-2
NGF
NGF
IFNIFN-γ γ
TNF-α
TNF-α
GM-CSF
GM-CSF
GH
GH
Origins
Origins
Production
Production（
（ g/L ） /L
B. licheniformis
B. cereus
B. halodurans
B. macerans
B. circulans
A. pernix
P. horikoshii
human
3.7
3.0
3
0.2
1.5
1.4
0.1
0.7
1.0
1
E. rhusiopathiae
T. pallidum
0.90.9
0.80.8
human
human
mouse
chicken
bovine
bovine
flounder
1.5
0.6
0.2
0.5
0.4
0.2
0.2
Table 1: Example of Proteins Expressed using the Brevibacillus
Expression System
Product Information
Brand
Product
Size
Cat.No
Takara
Brevibacillus Expression System II
1 Kit
HB200
Takara
Brevibacillus choshinensis Competent Cells
100 µL x 10
HB116
Takara
pNC-HisE DNA
10 µg
HB123
Takara
pNC-HisF DNA
10 µg
HB122
Takara
pNC-HisT DNA
10 µg
HB121
Takara
pNCMO2 DNA
10 µg
HB112
Takara
pNY326-BLA DNA
1 µg
HB114
Takara
pNI DNA
10 µg
HB131
Takara
pNI-His DNA
10 µg
HB132
Takara
pNY326 DNA
10 µg
HB111
6
E X P R E S S I O N
S Y S T E M S
Vectors for the Brevibacillus System
Lac operator
P2 promoter
sec signal peptide
His-Tag
MCS
ori−
ori＋
ColE1 ori
pNC-HisE
(5,263 bp)
Lac operator
P2 promoter
sec signal peptide
Lac operator
P2 promoter
sec signal peptide
His-Tag
MCS
ori−
ori＋
ColE1 ori
pNC-HisF
(5,260 bp)
rep
AmpR
pNC-HisT
(5,260 bp)
NmR
ori
＋
pNCMO2
(5.2 kb）
rep
Amp r
P2 promoter
ori –
pNI-His DNA
(5,079 bp)
rep
pNI DNA
(5,055 bp)
rep
P5 promoter
sec signal peptide
multiple cloning site
X-terminator
ori +
pNY326
(3.4 kp)
Nm r
rep
Ampr
Nmr
ori –
multi cloning site
ColE1 ori
NmR
All shuttle vectors between B.
choshinensis and E. coli contain the
P2 promoter, which is one of the five
promoters that control transcription of
the cell wall protein gene (HWP). This
promoter functions only in B. choshinensis
and not in E. coli, thereby ensuring robust
protein production only in B. choshinensis.
Lac operator
ori +
ColE1 ori
Ampr
NmR
ColE1 ori
Lac operator
His-Tag
multi cloning site
ori +
AmpR
AmpR
ori –
ori –
ori +
ColE1 ori
rep
P2 promoter
His-Tag
MCS
ori –
rep
Nm r
Nmr
The pNY326 vector* is maintained more stably than
pNC or pNI vectors in the host cells due to much
weaker promoter activity and smaller size (3.4
kb). The host strains containing the vector can be
repeatedly subcultured, may be used for scaled-up
production, and will continue to stably produce
protein. The pNY326 vector must be constructed by a
one-step method using B. choshinensis. Brevibacillus
choshinensis Competent Cells are used as the
transformation host.
*Can only be maintained in B. choshinensis
Choosing a Brevibacillus Vector?
Brand
Vector Name
Vector Type
Expression Vector
Lac
Operator
His-Tag
Sec Signal
Peptide
Construct in
Protease
cleavage site
X-terminator
Enterokinase
No
Takara
pNC-HisE (5,263 bp)
Shuttle Vector
Secretory
Yes
Yes
Yes
E. coli
Takara
pNC-HisT (5,260 bp)
Shuttle Vector
Secretory
Yes
Yes
Yes
E. coli
Thrombin
No
Takara
pNC-HisF (5,260 bp)
Shuttle Vector
Secretory
Yes
Yes
Yes
E. coli
Factor Xa
No
Takara
pNI-His DNA (5,079 bp)
Shuttle Vector
Intracellular
Yes
Yes
No
E. coli
Enterokinase
No
Takara
pNI DNA (5,055 bp)
Shuttle Vector
Intracellular
Yes
No
No
E. coli
No
No
Takara
pNY326 (3.4 Kb)
Expression
Secretory
No
No
Yes
Brevibacillus
No
Yes
Takara
pNCMO2 (5.2 kb)
Shuttle Vector
Secretory
No
No
Yes
E. coli
No
Yes
Takara
pNY326-BLA
Positive Control
Secretory
Includes a gene encoding Bacillus licheniformis a-amylase (55 kDa)
Application note
High-level Secretion of Recombinant Protein using the Brevibacillus Expression System
By Michikazu Tanio and Toshiyuki Kohno, Mitsubishi Kagaku Institute of Life Sciences (MITILS), Tokyo, Japan
www.XXXXX
7
E X P R E S S I O N
S Y S T E M S
BacPAK™ Baculovirus Expression System
The BacPAK Baculovirus Expression System expresses
recombinant proteins at extremely high levels (1 to 500 mg
of protein per liter of culture) in insect host cells (1, 2). The
BacPAK System offers three major advantages:
•High yield of recombinant protein. The insect host cells
produce large amounts of your target protein.
•Greater similarity to naturally occurring proteins.
The expressed recombinant protein is usually similar in
structure, biological activity, and immunological reactivity
to the naturally occurring protein because insect host cells
provide post-translational processing similar to that of
mammalian cells.
•High recombination efficiency. More than 90% of the
viruses produced by the transfected cells carry the target
protein. The specially designed BacPAK6 Viral DNA
forces recombination between the virus and transfer vector,
resulting in high recombination efficiency.
BacPAK Method
The target gene is inserted into a shuttle vector, which
is cotransfected into insect host cells with the linearized
BacPAK6 Viral DNA. The BacPAK6 DNA is missing an
essential portion of the baculovirus genome. When the DNA
recombines with the vector, the essential element is restored
and the target gene is transferred to the baculovirus genome.
Following recombination, a few viral plaques are picked and
purified, and the recombinant phenotype is verified. The
newly isolated recombinant virus can then be amplified and
used to infect insect cell cultures to produce large amounts of
the desired protein.
References
1. Kitts, P. A. & Possee, R. D. (1993) Biotechniques 15(5):810–817.
2. Kitts, P. A. et al. (1990) Nucleic Acids Res. 18:5667–5672.
In-Fusion® Ready BacPAK™ Vector Set
•In-Fusion technology greatly simplifies cloning
•Add N- or C-terminal polyhistidine tags
•Obtain high protein purity using TALON® resins
Baculovirus expression offers a significant advantage over
bacterial expression for generating large amounts of a
recombinant protein, since the posttranslational processing
and folding of recombinant proteins produced in insect cells
closely resembles mammalian processing and the yields of
functional protein are often much greater. The In-Fusion
Ready BacPAK Vector Set allows proteins to be quickly and
easily overexpressed in insect cells using In-Fusion cloning
technology, and efficiently purified using TALON Resin.
In-Fusion Cloning Simplifies Expression
In-Fusion cloning speeds the preparation of baculovirus
transfer vectors. It is simple, fast, accurate, directional, and
allows PCR products up to 12 kb in length to be directly
cloned without digestion or blunt-end polishing.
The In-Fusion Ready BacPAK vectors are prelinearized
and require no restriction enzyme digestion, phosphatase
treatment, or gel purification prior to cloning (1).
References
1. In-Fusion Ready BacPAK Vector Set (2006) Clontechniques XXI(2):16-17.
PCR product of your
gene of interest
with In-Fusion ends
AcMNPV
Sequence
pBacPAK
Nterm-6xHN
AcMNPV
Sequence
AcMNPV
Sequence
6xHN Stop
Poly A
AcMNPV
Sequence
pBacPAK
Cterm-6xHN
In-Fusion cloning
Baculovirus Accessory Products
• Saves time by shortening baculovirus expression experiments
up to six days
• Eliminates troublesome plaque assays
• Compatible with all commonly available (AcMNPV-based
baculovirus expression systems
The BacPAK Baculovirus Rapid Titer Kit provides a fast
and simple method for determining titers of baculovirus
stocks, typically the most time-consuming part of baculovirus
expression protocols. The kit uses a standard immunological
assay to accurately determine baculovirus titers within 48
ATG
Ppolyhedrin
andVector
transformation
The In-Fusion Ready BacPAK
Set and Baculovirus Expression
into E. coli
System. A PCR fragment containing your gene of interest is
simultaneously and directly cloned into the In-Fusion Ready BacPAK
Miniprep and sequence clones
Linear
Linear
Vector BacPAK6
pair
to generate
N- and C-terminal 6xHN-tagged
constructs.
BacPAK6
Cotransfect pBacPAK plasmid and BacPAK6
DNA
BacPAK™ Baculovirus Rapid Titer Kit
Stop
Poly A
ATG 6xHN
Ppolyhedrin
linear DNA into Sf9 or Sf21 insect cells
DNA
Sf9 or Sf21 insect cells
Harvest culture supernatant and amplify virus
Conduct small scale analysis
Large scale infection and expression
of recombinant protein
hours, whereas other methods, such as plaque and end-point
Purify by using TALON resin
dilution assays, require 4–8 days.
In the BacPAK Baculovirus Expression Systems, infected cells
express viral antigens long before plaques are formed. Therefore,
the Bac-PAK Rapid Titer assay allows titer determination after
a much shorter incubation period than traditional plaque assays
(1). Furthermore, the titers obtained with the Rapid Titer assay
are comparable to those obtained with other methods. This
kit is suitable for use with any virus stock with a titer of more
than 104 pfu/ml and is compatible with all commonly available
(AcMNPV-based) baculovirus expression systems.
References
1. Wolkman, L. E. & Goldsmith, P. A. (1982) Appl. Envir. Microbiol. 44 (1):227-233.
8
E X P R E S S I O N
• Determine viral titers in 4 hours with this rapid titration kit
• Harvest, titer and infect in a single day
• Suitable for any AcMNPV-type baculovirus
The BacPAK qPCR Titration Kit provides an extremely fast
and simple method for titrating your viral stocks. The kits
use a quick DNA purification step before determining viral
genome content using qPCR and SYBR® technologies (Figure
1). Whereas standard titration methods require up to 10 days
to complete, this titration kit requires only 4 hours and works
with any AcMNPV-based baculoviral vectors. Using qPCR
dramatically shortens the time interval between viral harvest
and target cell infection, allowing you to perform both on the
same day. This means that you can avoid delays that lead to
reduced viral infectivity and can infect target cells at a known
multiplicity of infection (MOI) for more consistent results
(1).
The procedure is simple: viral DNA and BacPAK control DNA
are serially diluted and subjected to qPCR.
The DNA copy number of each viral sample is then
determined by comparing its Ct value to a standard curve
generated by plotting the Ct values of the diluted control
samples against their respective copy numbers, as shown
in Figure 2. With its simplicity, reproducibility, and short
processing time, the BacPAK qPCR Titration Kit is ideal for
determining baculoviral DNA titers.
References
1. Rapid & Accurate Baculovirus Titration (2009) Clontechniques XXIV(3):8–9.
A
100
Fluorescence (dRn)
BacPAK™ qPCR Titration Kit
S Y S T E M S
The BacPAK qPCR Titration Kit allows you to determine the
viral genome copy number in baculoviral preparations from a
calibrated DNA standard curve (Figure 2).
10–1
10–2
0
5
10
15
B
20
Cycle no.
25
30
35
40
24
22
20
Ct (dRn)
18
Harvest baculoviral
supernatant
Purify viral DNA
Perform
qPCR
16
14
12
Analyze data
10
8
Figure 1. The BacPAK qPCR Titration Kit protocol.
6
104
105
106
Initial quantity (copies)
107
108
Figure 2. The BacPAK qPCR
Titration Kit exhibits a wide
dynamic range. The BacPAK
DNA Control Template was
serially diluted from 108 to 103
copies per sample and analyzed with the BacPAK qPCR
Titration Kit. The amplification
plots (Panel A) show a wide
dynamic range of at least 6
orders of magnitude with no
NTC (No-Template Control)
background.
The standard curve (Panel B)
obtained by plotting the Ct
values (determined from the
amplification plots in Panel
A) against the log of the DNA
copy number in each sample,
demonstrates a strong linear
correlation between the Ct and
the DNA copy number (log
scale), with R2 = 1.000 and a
PCR efficiency of 100%.
Comparison of BacPAK qPCR Titration to Other Titration Methods*
Titration Method
Plaque Assay
BacPAK Rapid Titer Assay
BacPAK qPCR Titration
Description
Count cleared plaques
in infected cell monolayer
Immunostaining of Gp64
in infected cell monolayer
Measure viral DNA using SYBR
qPCR with standard DNA as control
Time to Completion
1 week
48 hr
2–4 hr
Benefits
Traditional, visual
Simple, visual
Fast, accurate
* Clontech offers two different kits for baculovirus titration: the BacPAK Baculovirus Rapid Titer Kit (Cat. No. 631406) utilizes a standard
immunological assay to accurately identify virus-infected cells, and the BacPAK qPCR Titration Kit (Cat. No. 631414) measures viral DNA
copies via SYBR qPCR.
Product Information
Brand
Product
Size
Cat.No
Clontech
BacPAK Baculovirus Expression System
each
631402
Clontech
BacPAK6 DNA (Bsu36 I digest)
5 transfxns
631401
Clontech
IPLB-Sf21 Insect Cells
1 vial
631411
Clontech
BacPAK Grace’s Basic Medium
500 ml
631404
Clontech
X-GLUC
100 mg
631721
Clontech
In-Fusion Ready BacPAK Vector Set
3 vectors
631410
Clontech
In-Fusion® HD Cloning System
50 rxns
639646
Clontech
BacPAK Baculovirus Rapid Titer Kit
5 assays
631406
Clontech
BacPAK qPCR Titration Kit
200 rxns
631414
9
I N C R E A S E
P R O T E I N
Y I E L D
A N D
P U R I T Y
E.Coli
i n
pCold Expression Vectors
Features
• Great for difficult proteins that can’t be expressed with the T7 system
• Facilitates increased solubility due to expression at reduced temperature
• Facilitates increased purity due to repressed expression of host proteins
Application
• High-efficiency protein expression using a cold shock
promoter
Description
Takara’s pCold Expression Vectors offer Cold Shock
expression technology for high purity, high yield protein
production. The pCold series includes four different vectors.
Each includes the Cold Shock Protein A (cspA) promoter
for expression of highly pure recombinant protein in E. coli
at high yield. These vectors selectively induce target protein
synthesis at low temperature (15°C), a condition at which
host protein synthesis is suppressed and protease activity
is decreased. This results in high yields of target protein
(~60% of intracellular protein). In addition to the cspA
promoter, all four vectors contain a lac operator (for control
of expression), ampicillin resistance gene (ampr), ColE1
origin of replication, M13 IG fragment, and multiple
cloning site (MCS). Three vectors also contain either a
translation enhancing element (TEE), His-Tag sequence,
and/or Factor Xa cleavage site. These vectors work equally
well for synthesis of non-labeled and radiolabeled proteins
and can be used with Takara’s Chaperone Plasmid Set
(Cat. #3340).
Reference
1. Quing, G., et. al. (2004) Nature Biotechnol. 22(7):877-882.
Takara’s pCold expression vectors
pCold I
M
pCold II
ColE1 ori
ColE1 ori
IG
13
cspA 3'UTR
multiple cloning site
cspA 5'UTR
lac operator
cspA promoter
M
pCold III
4.4kb
IG
13
pCold IV
Amp
4.4kb
Amp
Amp
Amp
4.4kb
IG
13
cspA 3'UTR
multiple cloning site
TEE
cspA 5'UTR
lac operator
cspA promoter
lacI
M
lacI
IG
13
lacI
M
cspA 3'UTR
multiple cloning site
His•Tag
TEE
cspA 5'UTR
lac operator
cspA promoter
ColE1 ori
4.4kb
lacI
cspA 3'UTR
multiple cloning site
Factor Xa site
His•Tag
TEE
cspA 5'UTR
lac operator
cspA promoter
ColE1 ori
In the following examples, genes that were poorly expressed or that produced insoluble protein with the T7 promoter expression system were
expressed using the pCold system. pCold I DNA was used as an expression vector in E. coli. Expression from T7 promoter-driven vectors was
induced with IPTG and T7 plasmid-containing cells were cultured at 37°C.
N.C T7 pCold
T7
kDa
T
S
pCold
T
S
kDa
T: Entire protein fraction
S: Soluble fraction
97.4
97.4
66.2
← Expression level increased
66.2
45
45
← Expression enabled
31
21.5
31
21.5
14.4
14.4
CBB staining
CBB staining of the entire protein fraction
Expression of human gene A. Human gene A (~31 kDa) was
expressed in both the T7 system and the cold-shock expression
system. No expression was observed in the T7 system, but human
gene A was expressed in the pCold system.
Expression of human gene C. Comparison of expression of soluble
human gene C protein (~80 kDa) in the cold-shock expression system vs. the T7 system was performed. Target protein in the soluble
fraction of pCold cells was dramatically higher than that of the T7
system.
Product Information
Brand
Product
Size
Cat.No
Takara
pCold Vector Set
1 Set (ea.5 µg)
3360
Takara
pCold I DNA
25 µg
3361
Takara
pCold II DNA
25 µg
3362
Takara
pCold III DNA
25 µg
3363
Takara
pCold IV DNA
25 µg
3364
10
I N C R E A S E
P R O T E I N
Y I E L D
A N D
P U R I T Y
i n
E.Coli
pCold TF Vectors
Application
• Highly efficient protein expression using cold shock
technology
• High yield of active protein due to Trigger Factor chaperone
as a solubility-promoting fusion tag
Description
Takara’s pCold TF DNA Vector is a fusion cold shock
expression vector that expresses Trigger Factor (TF)
chaperone as a soluble fusion tag. Trigger Factor is a 45 kDa
prokaryotic ribosome-associated chaperone protein that
facilitates co-translational folding of nascent polypeptides.
Because of its E. coli origin, TF is highly expressed in
E. coli expression systems. The pCold TF DNA Vector
consists of the cspA promoter plus additional downstream
sequences including a 5’ untranslated region (5’ UTR), a
translation enhancing element (TEE), a His-Tag sequence,
and a multiple cloning site (MCS). A lac operator is inserted
downstream of the cspA promoter to ensure strict regulation
of expression. Additionally, recognition sites for HRV 3C
Protease, Thrombin, and Factor Xa are between TF-Taq and
the Multiple Cloning Site (MCS).
These sequences facilitate tag removal from the expressed
fusion protein. Most E. coli strains can serve as expression
hosts.
kDa
pCold TF
1 2
pCold
1 2
pCold +
Chaperone
1 2
T7
1 2
1. Cell extract solution
2. Soluble fraction
target protein
97
66
*
45
* co-expressed
trigger factor
31
22
Expression of protein A in T7 and pCold systems.
The expression of enzyme protein A (~29 kDa) was poor when
utilizing aT7 or pCold I expression systems, even when the pCold
I construct was co-expressed with a chaperone. In contrast, the
expression of target protein as a fusion (29 kDa + 52 kDa) was
successful with pCold TF DNA, and most of the expressed protein
was in soluble form. The expressed enzyme protein A showed
activity even in the form of a fusion protein (data not shown).
pCold ProS2 DNA
Features
Description
• Facilitates high yield protein expression with optimized protein folding
The pCold Pro S2 expression vector features Protein S, a
soluble tag from Myxococcus xanthus fused to the N-terminus
of target proteins. Tight regulation of protein expression is
maintained by a lac operator downstream of the cold shock
promoter. HRV 3C Protease, Thrombin, and Factor Xa
recognition sites are encoded between the Protein S tag and
the MSC to facilitate tag removal.
• Enables expression of fusion proteins with a soluble tag to optimize solubility
mRNA Interferase™- MazF Enzyme
Application
• Site-dependent cleavage of ssRNA
Description
MazF is a toxin protein in the toxin-antitoxin module of
E. coli. It possesses endoribonuclease activity and specifically
cleaves single-stranded RNA at the 5’ end of 5’-ACA-3’
sequences.
This enzyme does not cleave double-stranded RNA, doublestranded DNA, or single-stranded DNA. mRNA InterferaseMazF is supplied as a fusion protein of E. coli MazF and
Trigger Factor. The Trigger Factor protein is an E. coli
chaperone protein. The enzyme is also supplied with a 5X
MazF buffer (200 mM Sodium phosphate, pH 7.5, 0.05%
Tween 20.)
Product Information
Brand
Product
Size
Cat.No
Takara
pCold TF DNA
25 µg
3365
Takara
pCold™ ProS2 DNA
25 µg
3371
Takara
mRNA Interferase™-MazF
1000 units
2415A
Application note
The pCold TF Protein Expression System Produces Soluble, Active Protein in E. coli
www.XXXXX
11
I N C R E A S E
P R O T E I N
Y I E L D
A N D
P U R I T Y
i n
E.Coli
SPP System
Application
• Preferential expression of target protein by suppression of
endogenous proteins using mRNA interferase plasmid
Description
This system utilizes an E. coli protein, MazF, described as
an mRNA interferase by Suzuki et. al. MazF is a sequencespecific endoribonuclease that cleaves single strand RNAs
at 5’-ACA-3’ (ACA) sequences. When using the SPP
System, the transcript of interest should therefore lack ACA
sequences. MazF is co-expressed in the E. coli host and
suppresses expression of non-target genes by cleaving host
transcripts at ACA sequences. Therefore, the target protein
is the most abundantly expressed protein (Figure 1). Because
of the requirement for target gene transcripts to lack ACA
sequences, the SPP System is not suitable for all genes of
interest; however, when appropriate, it can result in extremely
high levels of protein production.
Figure 1. Synthesis of cspA-promoter expressed envAB in
presence and absence of MazF. E. coli BL21 cells co-expressing
MazF and pCold (SP-4) envZB showed good envZB expression
and extremely low background synthesis of host proteins.
Reference
Suzuki, M., et. al. (2005) Molecular Cell 18(2)253-261.
Product Information
Brand
Product
Size
Cat.No
Takara
SPP SystemTM I
1 kit
3367
Takara
SPP System II
1 kit
3368
Takara
SPP SystemTM III
1 kit
3369
Takara
SPP SystemTM IV
1 kit
3370
Takara
SPP SystemTM I-IV
1 kit
3366
TM
12
M A M M A L I A N
E X P R E S S I O N
V E C T O R S
A N D
S Y S T E M S
Human Cell-Free Expression System
Features
• Easy-to-use system allows generation of protein in as little as 1 h in a single-tube reaction
• Provides higher yield of functional protein and greater consistency than rabbit reticulocyte or wheat germ in vitro translation systems
• Excellent with challenging proteins such as large proteins (over 150 kDa) and proteins requiring post-translational modification
• Amenable to high-throughput screening; bulk sizes available.
Applications
• Expression of toxic proteins that are lethal to host cells of in
vivo expression systems
expressed using in vivo systems due to toxicity. In contrast to
expression using prokaryotic host cells, in vitro translation
systems also allow post-translational modifications such as
glycosylation, phosphorylation, and fatty acylation.
polyA
T7 terminator
Multiple Cloning Site
Factor Xa Site
His-Tag
EMCV
IRES
Ampr
pT7-IRES His-N DNA
(3,429 bp)
• Rapid analysis of protein function
• Rapid analysis of mutation series or truncation series:
generate protein quickly and assess functionality using your
downstream assay
ColE1 ori
Map of pT7-IRES vector.
• High-throughput proteomic studies
pT7-IRES Vector Information
• Expression of proteins that are easily degraded or insoluble in
conventional in vivo expression systems such as E. coli
Vectors in the pT7-IRES series include a T7 promoter
and EMCV IRES sequence to facilitate transcription and
translation in the Human Cell-Free Protein Expression
System, as well as a Multiple Cloning Site (MCS), convenient
tags (N- or C-terminal His-tag or Myc tag sequences), a
Factor Xa protease cleavage site for tag removal, poly-A
signal, and T7 terminator.
Description
The Human Cell-Free Protein Expression System from Takara
Bio is easy to use. The single-tube reaction is easily assembled
and protein synthesis is complete in as little as 1 h at 32°C.
The Human Cell-Free Protein Expression System provides
high yield (e.g.,
50 µg/ml of human eIF4G) of functional protein, including
proteins requiring modifications such as glycosylation,
phosphorylation, or fatty acylation. Excellent yield is observed
even with large proteins (over 150 kDa). Genes of interest
may be cloned rapidly into pT7-IRES vectors using InFusion cloning technology. After expression, proteins with
N-terminal or C-terminal His-tag or N-terminal Myc tag
can be generated, depending on choice of vector. Bulk sizes
are available for high-throughput studies; contact custom@
takara-bio.eu for more information.
In vitro translation has many advantages for protein
expression: it is excellent for rapid studies of protein function
or features, amenable to high-throughput studies, useful for
proteins that are degraded or insoluble with in vivo systems,
and can be used to generate lethal proteins that cannot be
• pT7-IRES His-N DNA (Cat. # 3290) encodes an
N-terminal His-tag
• pT7-IRES His-C DNA (Cat. # 3291) encodes a C-terminal
His-tag
• pT7-IRES Myc-N DNA (Cat. # 3292) encodes an
N-terminal c-Myc tag
Related In-Fusion Cloning Products
For rapid and easy cloning, use the In-Fusion HD Cloning
System (Clontech Cat. # 639645/ 639646/639692/ 639647)
or In-Fusion HD Cloning System CE (Clontech Cat. #
639636/ 639637/639693/ 639638) to generate pT7-IRES
constructs with your insert of interest.
13
M A M M A L I A N
E X P R E S S I O N
V E C T O R S
A N D
S Y S T E M S
Principle of the Human Cell-Free Protein Expression System.
A) In an easy and simple protocol, target protein translation is initiated by adding pT7-IRES Vector containing the target gene cassette and
other kit components. B) The target gene RNA transcribed from the pT7-IRES Vector has an IRES sequence designed to promote protein
translation initiation. As protein synthesis progresses, the translation initiation factor from the cell lysate becomes inactivated. The translation
enhancement factor in the reaction mixture, however, reactivates this inactivated translation initiation factor and thereby maintains a high
level of translation.
1
Relative absorbance
120
2
3
100
80
Mixture-2(＋)
Mixture-2(ー)
60
40
20
0
0
1
2
3
4
5
6
7
8
◄◄
Time (hour)
Time course of β-galactosidase expression and effect of translation
enhancement factor. Eight replicates of β-galactosidase in vitro
translation were performed using 1 µL of Control Vector per
reaction. At each of the indicated time points (0, 0.5, 1, 2, 3, 4.5, 6,
and 8 h after start of the reaction), one reaction tube was removed
and used for β-galactosidase activity assay with O-nitrophenylβ-D-galactopyranoside (ONPG) as the substrate. A separate
set of reactions were conducted in absence of the translation
enhancement factor (Mixture-2). The activity of β-galactosidase
increased over time to peak at approximately 4.5 h. Additionally,
the presence of Mixture-2 containing translation enhancement
factor markedly increased yield of active protein.
Synthesis of high molecular weight proteins
using the Human Cell-Free Protein Expression
System. In vitro translation reactions were
performed to synthesize human Dicer (200 kDa,
lane 2) or human eIF4G (170 kDa, lane 3) protein.
Reactions were analyzed by SDS-PAGE and
Coomassie blue staining. Arrowheads indicate
target proteins. Lane 1, negative control.
Product Information
Brand
Product
Size
Cat.No
Takara
Human Cell-Free Protein Expression System
10 rxns
3281
Takara
pT7-IRES His-N DNA
20 µg
3290
Takara
pT7-IRES His-C DNA
20 µg
3291
Takara
pT7-IRES Myc-N DNA
20 µg
3292
14
C L O N I N G
M E T H O D S
Optimal PCR-cloning method for protein expression
A simple 15-30 minute In-Fusion® reaction results in the creation
of seamless and precisely engineered constructs, where no extra bp
of vector or restriction-site-derived DNA is added. This is a crucial
advantage when generating constructs to express tagged proteins
as no undesirable vector or restriction-site-derived amino acids are
added to the expressed protein, unless specifically required by you.
This allows the expressed tagged protein to mimic the native
protein as closely as possible, in order to aid its correct folding,
especially important in studies of protein function.
Single-tube protocol
x
x
Recombinant
vector
Amplify your
gene of interest
PCR
product
Gene-specific primers
with 15 bp extensions
homologous to vector ends
The In-Fusion enzyme
produces single-stranded
PCR fragment and vector ends
that are fused due to the
15 bp homology
Any
linearized
vector
In-Fusion allows seamless, directional cloning of PCR products directly into your vector of choice and generates more
than 90% correct clones.
Product name
Cells included?
Cloning Enhancer
included?
Nucleospin
columns included?
In-Fusion® HD Cloning
Kit
Liquid
format
Dry-down
format
In-Fusion® HD Cloning
System CE
Yes
In-Fusion® HD Cloning
System
Yes
Yes
Yes
In-Fusion® HD EcoDry™
Cloning Kit
In-Fusion® HD EcoDry™
Cloning System
Yes
Yes
Size (rxns)
Cat. No.
10; 50; 100
639648; 639649;
639650
10; 50; 100
639636; 639637;
639638
10; 50; 100
639645; 639646;
639647
8; 24; 96
639689; 639690;
639691
8; 24; 48; 96
639684; 639685;
639686; 639688
For complete list of In-Fusion products available please see www.clontech.com
PCR enzymes perfect for In-Fusion® cloning!
In-Fusion cloning can be performed with inserts generated by
any PCR enzyme independent of whether they are sticky or blunt
ended PCR products. Clontech’s Advantage HD is a high fidelity
enzyme suitable also for use with small amounts of template
DNA. Takara’sPrimeSTAR enzymes are also recommended for use
with In-Fusion due to their capacity of combining high fidelity
with long and GCrich targets or speed.
PrimeSTAR GXL is the best commercially available enzyme
for amplifyinglong targets (up to 30 kb) and can be used with
excess as well as GC/AT-rich template DNA Takara’s high fidelity
andhigh speed enzyme, PrimeSTAR Max, has the fastest extension
speed available (≤ 5 sec./kb), along with extremely high accuracy,
specificity and sensitivity.
Product Information
Brand
Product
Size
Cat.No
Takara
Advantage HD
200 rxns
639241
Takara
PrimeSTAR GXL DNA Polymerase
250 Units
R050A
1000 Units
R050B
100 rxns
R045A
400 rxns
R045B
Takara
PrimeSTAR Max DNA Polymerase
15
Brand
Vector
Type
Expression
Tag/marker
Cleavage
Size
E. Coli Expression vectors
Clontech
pET6xHN-N
bacterial
Constitutive
6xHN
enterokinase
5.8
Clontech
pET6xHN-C
bacterial
Constitutive
6xHN
thrombin
5.8
Clontech
pET6xHN-GFPuv
bacterial
Constitutive
N-terminal 6xHN
enterokinase
6.5
Clontech
pET6xHN-N
bacterial
Constitutive
6xHN
enterokinase
5.8
Clontech
pET6xHN-C
bacterial
Constitutive
6xHN
thrombin
5.8
Clontech
pEcoli-6xHN-GFPuv
bacterial
Constitutive
N-terminal 6xHN
enterokinase
6.5
Baculovirus Expression vectors
Clontech
In-Fusion Ready pBacPAK-Cterm 6xHN
insect cells
Constitutive
6xHN
thrombin
5.4
Clontech
In-Fusion Ready pBacPAK-Nterm 6xHN
insect cells
Constitutive
6xHN
enterokinase
5.4
Clontech
pBacPAK8
insect cells
Constitutive
None
None
5.5
Clontech
pBacPAK9
insect cells
Constitutive
None
None
5.5
Mammalian Expression vectors
Takara
pBApo-CMV Neo DNA (also for miRNA, adeno)
mammalian
Constitutive
None
Takara
pBApo-CMV Pur DNA (also for miRNA, adeno)
mammalian
Constitutive
None
Takara
pBApo-CMV DNA (also for miRNA, adeno)
mammalian
Constitutive
None
Takara
pBApo-EF1alpha Neo DNA (also for miRNA, adeno)
mammalian
Constitutive
None
Takara
pBApo-EF1alpha Pur DNA (also for miRNA, adeno)
mammalian
Constitutive
None
Clontech
pCMV-HA-C
mammalian
Constitutive
HA epitope
3.8
Clontech
pCMV-HA-N
mammalian
Constitutive
HA epitope
3.8
Clontech
pCMV-Myc-C
mammalian
Constitutive
myc epitope
3.8
Clontech
pCMV-Myc-N
mammalian
Constitutive
myc epitope
3.8
Clontech
pEF1-Alpha-Myc
mammalian
Constitutive
myc epitope
None
4.6
Clontech
pEF1-Alpha-HA
mammalian
Constitutive
HA epitope
None
4.6
Clontech
pCMV-(DYKDDDDK)-C
mammalian
Constitutive
DYKDDDDK (FLAG) epitope:
3.8
Clontech
pCMV-(DYKDDDDK)-N
mammalian
Constitutive
DYKDDDDK (FLAG) epitope:
3.8
Clontech
Fluorescent Protein Vectors - fusion
mammalian
Constitutive
Fluorescent Protein
4.7
Clontech
mammalian
Constitutive
Fluorescent Protein
5.5
Clontech
lentiviral
Constitutive
Fluorescent Protein
8,8; 9,5
Clontech
lentiviral
Constitutive
Fluorescent Protein
9.5
mammalian
Constitutive
Fluorescent Protein
5.3
Clontech
mammalian
Constitutive
Fluorescent Protein
6
Clontech
lentiviral
Constitutive
Fluorescent Protein
8,2; 8,9
Clontech
lentiviral
Constitutive
Fluorescent Protein
8.9
Clontech
Fluorescent Protein Vectors - bicistronic
Inducible Mammalian Expression Systems
Clontech
Tet-ON 3G
mammalian
Inducible
None
None
3.4
Clontech
Tet-OFF Advanced
mammalian
Inducible
None
None
2.6
Clontech
Tet-On 3G - EF1-Alpha Version
mammalian
Inducible
None
None
3.4
Clontech
Tet-On 3G - Bidirectional
mammalian
Inducible
with or without fluorescent
proteins
2,9; 3,6
Clontech
Tet-On 3G - Bicistronic
mammalian
Inducible
with or without fluorescent
proteins
4; 4,7
Clontech
Tet-On 3G - Lentiviral: pLVX-TRE3G
lentiviral
Inducible
None
None
7.8
Clontech
Tet-On 3G - Retroviral: pRetroX-TRE3G
retroviral
Inducible
None
None
6.6
Clontech
Tet-On 3G - Adenoviral: pAdenoX-Tet3G
adenoviral
Inducible
None
None
36
Clontech
Tet-Express System
mammalian,
lentiviral,
retroviral
Inducible
with or without fluorescent
proteins
None
16
Promoter
Selection
Prok.
Selection
Euk.
MCS
replication
origin
Sold as part of
single cat no
T7 lac
Ampr
N/A
10
pBR322
low copy
T7 lac
Ampr
N/A
10
pBR322
low copy
not sold separately
T7 lac
Ampr
N/A
pBR322
low copy
not sold separately
T7 lac
Ampr
N/A
In-Fusion
Ready
pBR322
low copy
T7 lac
Ampr
N/A
In-Fusion
Ready
pBR322
low copy
T7 lac
Ampr
N/A
pBR322
low copy
Polyhedrin
Ampr
N/A
In-Fusion
Ready
pUC
high copy
631410
Polyhedrin
Ampr
N/A
In-Fusion
Ready
pUC
high copy
631410
Polyhedrin
Ampr
N/A
18
pUC
high copy
631402
Polyhedrin
Ampr
N/A
18
pUC
high copy
631402
CMV IE
Ampr
Neor
9
high copy
3240
CMV IE
Ampr
Puror
9
high copy
3241
CMV IE
Ampr
None
13
high copy
3242
EF-1a
Ampr
Neor
9
high copy
3243
EF-1a
Ampr
Puror
9
high copy
3244
CMV IE
Ampr
None
7
pUC
high copy
635690
CMV IE
Ampr
None
7
pUC
high copy
631604, 635690
CMV IE
Ampr
None
7
pUC
high copy
635689
CMV IE
Ampr
None
7
pUC
high copy
635689, 631604
EF-1a
Ampr
None
8
pUC
high copy
631991
EF-1a
Ampr
None
9
pUC
high copy
631992
CMV
Ampr
None
7
pUC
high copy
635688
CMV
Ampr
None
12
pUC
high copy
635688
CMV IE
Kanr
Neor
pUC
high copy
several refs
EF-1a
Kanr
Neor
pUC
high copy
several refs
CMV IE
Ampr
Puror
7
pUC
high copy
several refs
EF-1a
Ampr
Puror
5
pUC
high copy
severals refs
CMV IE
Kanr
Neor
13
pUC
high copy
632540, 632435, 632420,
632478
EF-1a
Kanr
Neor
6; 8
pUC
high copy
631971, 631980, 631976
CMV IE
Ampr
None
6
pUC
high copy
632187, 631237, 631238
EF-1a
Ampr
None
5
pUC
high copy
631982, 631987
TRE3G
Ampr
None
11
pUC
high copy
Tight
Ampr
None
15
Col E1
low copy
TRE3G
Ampr
None
11
pUC
high copy
631167
not sold separately
TRE3G-BI
Ampr
None
pUC
high copy
631337, 631340, 631338,
631341, 631339,631342
not sold separately
TRE3G
Ampr
None
pUC
high copy
631164, 631165, 631166,
631346, 631347, 631347
not sold separately
TRE3GV
Ampr
None
8
pUC
high copy
631187
not sold separately
TREGV
Ampr
Puror
7
pUC
high copy
631188
not sold separately
TRE3G
Ampr
None
In-Fusion
Ready
pUC
high copy
631180
not sold separately
TRE3G,
TRE3G-BI,
TRE3GV
Ampr
None
pUC
high copy
631169, 631343, 631170,
631344, 631345, 631171,
631172, 631189, 631190
not sold separately
631431, 631430,
Available as single
item
not sold separately
631429,0631428,
not sold separately
not sold separately
631417
631168
not sold separately
631059
17
F O L D I N G
Chaperone Plasmid Set
Application
• Promotes correct in vivo folding of expressed recombinant
proteins in E. coli
Plasmids with the Chaperone Plasmid Set work well in
combination with the pCold expression system vectors.
araC
Description
The Chaperone Plasmid Set consists of 5 different plasmids,
each of which is designed to express multiple molecular
chaperones. Together, they function as a “chaperone team”
to facilitate protein folding. Co-expression of a target protein
with one of these plasmids increases the recovery of soluble
proteins. Each plasmid carries an origin of replication derived
from pACYC and a Cmr gene, which allows use with E. coli
expression systems utilizing ColE1-type plasmids with an
ampicillin resistance gene as a marker. The chaperone genes
are situated downstream of an araB or Pzt-1 (tet) promoter.
Therefore, expression of target proteins and chaperones can
be induced individually if the target gene is placed under
the control of other promoters (e.g. lac). These plasmids
also contain the necessary regulator (araC or tetR) for each
promoter.
Note that this system cannot be used in combination with
chloramphenicol-resistant E. coli host strains or expression
plasmids that carry a chloramphenicol-resistance gene.
For example, E. coli BL21(DE3), which is often used with
pET systems, is a compatible host strain. However, E. coli
BL21(DE3) pLysS and BL21(DE3) pLysE, which contain
pLysS or pLysE plasmids that have the pACYC replication
origin and the Cmr gene, cannot be used with this system.
Human gene A (~70 kDa) was expressed
in insoluble form when using pCold
I alone. However, the level of soluble
expressed protein increased significantly
when the chaperone plasmid pG-Tf2 was
co-expressed with the pCold I construct.
dnaK
Cmr
Cmr
groEL
araB
pG-KJE8
11.1 kb
araC
groES
Pzt1
5.4 kb
tetR
dnaJ
Cmr
araB
araB
groES
pACYC ori
grpE
groEL
Pzt1
rrnBT1T2
araB
gro ES
pACYC ori
pACYC ori
araC
pACYC ori
pGro7
Cmr
pKJE7
pTf16
8.3 kb
grpE
dnaJ
tetR
tig
gro EL
pG-Tf2
7.2 kb
dnaK
araC
Cmr
5 kb
tig
pACYC ori
Resistance
Plasmid Chaperone
PromoterInducer
Marker
pG-KJE8 dnaK-dnaJ-grpE-groES-groEL araB, Pzt1 L-Arabinose, TetracyclineCmr
pGro7groES-groEL
araB
L-Arabinose
Cmr
pKJE7dnaK-dnaJ-grpE
araB
L-Arabinose
Cmr
pG-Tf2groES-groEL-tig
Pzt1
Tetracycline
Cmr
pTf16tig
araB
L-Arabinose
Cmr
References
1. Nishihara, K., et al. (2000) Microbiol. 66(3):884-889.
2. Nishihara, K., et al. (1998) Appl. Environ. Microbiol. 64(5):1694-1699.
Human gene B (~24 kDa) was not
expressed when using pCold I DNA
alone. However, co-expression with the
chaperone plasmid pG-Tf2 resulted in
expression of high levels of target protein
in soluble form.
The combination of Cold Shock Expression Vectors and the Chaperone Plasmid Set often leads to significant improvement in expression level of soluble forms of target proteins. If sufficient expression or solubilization cannot be achieved using pCold vectors alone, we
recommend co-expression with chaperone plasmids. Furthermore, pCold vector-based expression systems may produce better results
by co-expressing chaperone plasmids carrying the tig sequence, such as pG-Tf2 or pTf16, which are included in the Chaperone Plasmid
Set (data not shown).
Product Information
Brand
Product
Size
Cat.No
Takara
Chaperone Plasmid Set
1 Set
3340
18
F O L D I N G
Chaperonin GroE
Application
• Facilitates refolding of denatured proteins
Description
Chaperonin GroE is a protein complex composed of
GroEL (14 subunits, 57 kDa) and GroES (7 subunits, 10
kDa).
It is thought to support the ability of proteins to form
tertiary structure upon or immediately after translation.
GroE is essential to assembly (and presumably reassembly
after denaturation) of protein complexes in vivo. Chaperonin
GroEL and GroES can be used for refolding denatured
proteins to recover functional activity.
Corystein™ (Purothionin) Reagent
Application
Description
• Facilitates protein refolding by promoting exchange
reactions between disulfide bonds
Corystein™ (Purothionin) Reagent is a polypeptide purified from wheat
endosperm. It catalyzes the formation of correct disulfide bonds in pro­
teins. Corystein™ Reagent can be used alone or together with thioredoxin
on a variety of proteins to re-form disulfide bonds.
Refolding CA Kit
Application
Overnight incubation of the CA-treated protein is followed by a
quick 10-minute centrifugation. The resulting supernatant contains
the refolded protein.
• Refolding of isolated inclusion body proteins
Description
The Refolding CA Kit uses a novel artificial chaperone technology
(licensed from NFRI, BTRAI, and Ezaki Glico Co, Ltd.) in an
easy 2-step procedure for optimizing the refolding conditions of
inclusion body proteins. Optimization allows identification of
the best conditions for correct protein folding and restoration of
protein activity.
The Small Kit (Cat. #7350) is supplied with guanidine
hydrochloride and DTT for protein denaturation, four different
surfactants that can be added independently to the unfolded
protein solution to protect against molecular aggregation, and
highly polymerized cycloamylose (CA), an artificial chaperone, for
surfactant removal and recovery of protein activity.
The Large Kit (Cat. #7351) is used for large-scale refolding after
initial determination of with the Small Refolding CA kit, and
consists only of denaturant and CA.
References
1. Machida, S., et al. (2000) FEBS Lett. 486(2):131-135.
2. Sundari, C.S., et al. (1999) FEBS Lett. 443(2):215-219.
3. Daugherty, D.L., et al. (1988) J. Biol. Chem 273(51):33961-33971.
Application note
Unfolding the Potential of Proteins
New refolding technologies are essential for tomorrow’s
recombinant proteins, By Joby Marie Chesnick
www.XXXXX
Principle of the Refolding CA Kit
Guanidine
hydrochloride
unfolds inclusion
bodies
Surfactants
prevent
protein
aggregation
Highly polymerized CA
removes surfactants
and facilitates protein
refolding
Biologically
active protein in
thermodynamically
stable native
conformation
Product Information
Brand
Product
Size
Cat.No
Takara
Chaperonin Gro EL
5 mg
7330
Takara
Chaperonin Gro ES
0.5 mg
7331
Takara
Corystein™ (Purothionin) Reagent
5 mg
7311
Takara
Refolding CA Kit
25 reactions
7350 (small)
Takara
Refolding CA Kit
1 kit
7351 (large)
19
P R O T E I N
P U R I F I C AT I O N
Talon® Co Resin-Highest Purity
Advantages
Applications
• Highest purity
• Crystallography
• Highest specificity for his-tagged proteins
• Functional assays
• Reduced copurification of impurities
• Structural & functional investigations
TALON resin is designed to maximize your yield of
biologically active protein. Choose TALON when purity
is of utmost importance or when purifying under native
conditions.
0
0.02
0.01
0
10
20
Time (min)
Elute
Wash
Load
Elute
10
20
Time (min)
0.03
Contaminant peak
coelutes
0.01
Ni-NTA
0.04
A475 (AU)
0.02
Wash
Load
0.03
Contaminant peak
is separated
TALON
0.04
A475 (AU)
Each TALON reactive core is charged with a cobalt ion that
has a much higher selectivity for histidine tags than nickel
ions. Only proteins containing adjacent histidines or specially
positioned histidines are able to bind to TALON. In contrast,
the spatial requirement for nickel-based resins is less strict,
so using Ni-NTA resins often results in copurification of
contaminants (see figure).
30
40
IMAC purification—TALON vs. Ni-NTA. 6xHN-AcGFP1 was purified from
Sf21 cells using TALON or Ni-NTA. The chromatogram for each column
is shown. The absorbance at 475 nm indicates the amount of target
protein (AcGFP1) present in each fraction.
His60 SuperflowTM Resin-Highest Capacity
Advantages
Applications
• Highest binding capacity (60 mg/ml)
• Protein labeling
• Purify proteins under native or denaturing conditions
• Immunization to produce antibodies
• Low leakage of Ni2+ ions
• Animal studies
His60 Ni Superflow Resin is a high-capacity resin for the
efficient purification of his-tagged proteins. The resin enables
fast, easy, and reproducible chromatographic separations and can
be reused for the same protein. His60 Ni Resin is compatible
with batch/gravity-flow applications as well as with the major
automated liquid chromatography systems and manual syringe
processing.
In one application, His60 Ni Superflow Resin performance was
compared to the performance of Competitor Q’s Ni Superflow
resin. 6xHN-AcGFP1 was purified from equivalent amounts
of the same sample, following each manufacturer’s protocol.
Clontech’s pEcoli Linear Expression System was used to express
6xHN-AcGFP1 in E. coli. Higher yields & better purity were
obtained using His60 Ni Superflow Resin (see figure).
A
kD
His60 Ni Resin
M OS FT W1W2 E1 E2 E3 E4
150–
100–
75–
Higher
purity
Higher
yield
50–
37–
6xHN-AcGFP1
25–
20–
15–
B
kD
150–
100–
75–
NI-NTA Superflow Resin
M OS FT W1W2 E1 E2 E3 E4
Lower
purity
Lower
yield
50–
37–
25–
20–
15–
6xHN-AcGFP1
His60 Ni Superflow Outperforms Ni-NTA. Superior yields and better
purity were obtained when comparing His60 Ni Superflow (Panel A) to
Competitor Q’s Ni resin (Panel B).
20
P R O T E I N
P U R I F I C AT I O N
TALON Resin and Magnetic Beads
TALON Resin
• Use for batch/gravity applications
• Highest purity of your target protein
TALON Superflow Resin
• Use for FPLC applications
• Highest purity of your target protein
TALON CellThru Resin
• Purify proteins directly from crude cell lysates, sonicates, and fermentation liquids
• Ideal for purification of membrane-bound proteins and multiprotein complexes
TALON Magnetic Beads
• Microscale purification or screening of his-tagged proteins (bead particle size:
20–75 µm)
• Elute in small volumes (50–200 µl)
His60 Ni Resin
His60 Ni Superflow Resin
• Use for batch/gravity and FPLC applications
• Highest binding capacity—60 mg/ml
• Superior performance compared to other Ni-based resins
(highest capacity and better purity)
• Low nickel ion leakage
• Purify under native or denaturing conditions
• Many sizes are available, from 10 ml to 1,000 ml
Buffers and Detection Reagents
Buffers
• HisTALON Buffer Set: complete buffer set for extraction and purification
of his-tagged proteins using TALON resin
• His60 Ni Buffer Set: complete buffer set for extraction and purification
of his-tagged proteins using His60 Ni Superflow resin
• xTractor Buffer: optimized lysis buffer for recombinant protein extraction
• xTractor Buffer Kit: complete extraction kit with lysozyme and DNase I,
for efficient extraction of high molecular weight proteins
Detection of His-Tags
Antibodies
• 6xHis mAb-HRP conjugate
• 6xHis Monoclonal Antibody (Albumin Free)
• 6xHN Polyclonal Antibody
21
P R O T E I N
P U R I F I C AT I O N
1 ml & 5 ml Cartridges
HisTALON Superflow
His60 Ni Superflow
FPLC Cartridges—HisTALON™ & His60 Ni
Ready-to-use 1 ml and 5 ml cartridges
• Use on any chromatography system
• Simple manual operation using a syringe
• Can be connected in series to scale-up
Gravity Columns
HisTALON
His60 Ni Superflow
Gravity Columns—HisTALON & His60 Ni
Ready-to-use 1 ml columns
• Up to 60 mg pure his-tagged protein
per His60 Ni Superflow column
• Up to 20 mg highly pure his-tagged protein
per HisTALON column
TALON Single Step Columns
5 ml
20 ml
Single Step Columns—TALON Single Step
Ready-to-use 5 ml and 20 ml columns
• Load bacterial culture directly onto column
• On-column extraction and purification
• Load bacterial culture, wash, and elute target
protein
TALONSpin Columns
Disposable Spin Columns—TALONSpin™
Ready-to-use prepacked spin columns
for small-scale purification
• Rapid, parallel purification in as little as 30
minutes
• Applications: screening of positive clones,
trial-level purification
TALON 2 ml Disposable Gravity Columns
Empty 2 ml Disposable Columns—TALON
Ready-to-use empty 2 ml gravity columns
• Fill columns with TALON, TALON Superflow,
or His60 Ni Superflow resins
• Also suitable for glycoprotein enrichment,
phosphoprotein & phosphopeptide enrichment,
GST-tag, and antibody purification resins
CellThru 10 ml Disposable Columns
Empty 10 ml Disposable Columns—CellThru
Ready-to-use empty 10 ml gravity columns
• Fill columns with TALON CellThru, TALON,
TALON Superflow, or His60 Ni Superflow resins
• Also suitable for glycoprotein enrichment,
phosphoprotein & phosphopeptide enrichment,
and GST-tag & antibody purification resins
22
P R O T E I N
P U R I F I C AT I O N
Clontech Protein Purification Products Information
Product
Resine type
Size
Cat.No
Cobalt
10 ml
25 ml
100 ml
250 ml
2 x 250 ml
4 x 250 ml
635501
635502
635503
635504
635652
635653
TALON Superflow Metal Affinity Resin
Cobalt
25 ml
100 ml
250 ml
2 x 250 ml
4 x 250 ml
635506
635507
635670
635669
635668
TALON CellThru
Cobalt
10 ml
100 ml
635509
635510
TALON Magnetic Beads
Cobalt
2 x 1 ml
6 x 1 ml
635636
635637
HisTALON Gravity Columns
Cobalt
5 columns
635655
HisTALON Gravity Columns Purification Kit
Cobalt
20 purifications
635654
TALONspin Columns
Cobalt
10 columns
25 columns
50 columns
635601
635602
635603
TALON Single Step Columns (5 ml)
Cobalt
2 columns
25 columns
635631
635628
TALON Single Step Columns (20 ml)
Cobalt
10 columns
635632
HisTALON Superflow Cartridges (5 x 1 ml)
Cobalt
5 cartridges
635650
HisTALON Superflow Cartridge (1 x 5 ml)
Cobalt
1 cartridge
635683
HisTALON Superflow Cartridges (5 x 5 ml)
Cobalt
5 cartridges
635682
HisTALON Superflow Cartridge Purification Kit (5 x 1 ml)
Cobalt
20 purifications
635649
HisTALON Superflow Cartridge Purification Kit (5 x 5 ml)
Cobalt
5 purifications
635681
CellThru 10-ml Disposable Columns
Cobalt
20 columns
635513
TALON 2 ml Disposable Gravity Column
Cobalt
50 columns
635606
Nickel
10 ml
25 ml
4 x 25 ml
250 ml
2 x 250 ml
4 x 250 ml
635659
635660
635661
635662
635663
635664
His60 Ni Gravity Columns
Nickel
5 columns
635657
His60 Ni Gravity Columns Purification Kit
Nickel
20 purifications
635658
His60 Ni Superflow Cartridges (5 x 1 ml)
Nickel
5 cartridges
635675
His60 Ni Superflow Cartridge (1 x 5 ml)
Nickel
1 cartridge
635680
His60 Ni Superflow Cartridges (5 x 5 ml)
Nickel
5 cartridges
635679
His60 Ni Superflow Cartridge Purification Kit (5 x 1 ml)
Nickel
20 purifications
635674
His60 Ni Superflow Cartridge Purification Kit (5 x 5 ml)
Nickel
5 purifications
635678
TALON Resin
TALON Metal Affinity Resin
TALON Prepacked
TALON Disposable Columns
His60 Ni Superflow Resin
His60 Ni Superflow
His60 Ni Prepacked
23
P R O T E I N
P U R I F I C AT I O N
Protein Purification – other tags
Co
mp
ant etito
r
i-FL
AG S
bea
ds
Clo
nt
DY ech A
KD
n
DD tiDK
bea
ds
The FLAG tag is commonly known as the DYKDDDDK epitope
tag and is used as an N- or C- terminal epitope tag in many fusion
proteins because it is small and therefore unlikely to alter the
biochemical properties or localization of the protein to which it is
fused.
Clontech’s Anti-DYKDDDDK Beads and Immunoprecipitation
Buffer Set can be purchased separately to make a complete CoIP kit for purification of FLAG-tagged proteins.
Ce
ll ly
sat
e
DYKDDDDK (FLAG Epitope) Tagged Proteins:
Immunoprecipitation and Detection
The anti-DYKDDDDK Antibody can be used for the
detection of FLAG-tagged proteins. This monoclonal antibody
recognizes the well-known FLAG epitope tag and detects N- or
C-terminally tagged FLAG fusion proteins on Western blots. The
highly specific and sensitive antibody does not cross-react with
endogenous proteins.
Anti-DYKDDDDK Beads consist of a monoclonal antiDYKDDDDK antibody, crosslinked to agarose beads. These
beads are used to purify or immunoprecipitate DYKDDDDKtagged or FLAG-tagged fusion proteins from cell lysates.
Heavy chain
N-term
DYKDDDDK-AcGFP1
Light chain
Anti-DYKDDDDK Beads for immunoprecipitation provide higher
purity and lower background when compared with competitor
S anti-FLAG beads.
Product Information
Brand
Product
Size
Cat.No
Clontech
Anti-DYKDDDDK Antibody
200 µg
635691
Clontech
Anti-DYKDDDDK Beads
1 ml
635686
Clontech
Immunoprecipitation Buffer Set
30 rxns
635687
Clontech
pCMV-DYKDDDDK Vector Set
10 µg each
635688
GST-Tag Purification Resins
• One-step isolation of highly pure GST-tagged proteins
respectively, with glutathione covalently bound to the resins.
• High binding capacity (>10 mg tagged protein per ml resin)
Glutathione-Superflow Resin is suitable for FPLC applications.
It can withstand higher flow rates and back pressure with flow
rates as high as 15 ml/cm2/min. Alternatively, GlutathioneUniflow Resin, with a maximum linear flow rate of 2 cm2/min,
is suitable for purification of large fusion proteins using batch/
gravity-flow purification or standard chromatography methods.
Glutathione-Superflow and -Uniflow Resins bind GST
(glutathione-S-transferase) tags with high affinity and specificity,
allowing rapid, efficient purification of GST-tagged proteins.
These resins are based on 6% and 4% cross-linked agarose,
II
GST-DHFR
Elute
55
20
Wash
6.5
0.0
5
15
25
Effluent (ml)
Western
WC
205
II
I
55
Elute
I
kDa
1.0
Load
ELU
205
Absorbance (254 nm)
B
E
FT
WC
kDa
1.0
Load
Absorbance (254 nm)
A
For greater convenience, the GST Purification Kit provides
sufficient stock buffers and prepacked Glutathione-Uniflow
Columns for performing 5 batch/gravity-flow purifications. Up
to 10 mg of GST-tagged protein per column can be purified
using this kit.
ELU
Flexible Resin Formats
E
• Easily regenerated for reuse & competitively priced
FT
• Available in flexible formats for gravity flow & FPLC
applications
GST
Wash
20
6.5
0.0
4
8
10
20
30
Western
Effluent (ml)
Figure 1. GST-tagged protein purification from whole cell extract. Whole cell extracts containing GST-DHFR (Panel A) and GST alone (Panel
B) were loaded, washed, and eluted from glutathione resin columns. The resulting purification fractions were analyzed by SDS-PAGE (upper
panels) and Western blot (lower panels) with an anti-GST IgG. WCE = whole cell extract. FT = flowthrough. ELU = eluate.
Product Information
Brand
Product
Size
Cat.No
Clontech
Glutathione-Superflow Resin
10 ml
635607
Clontech
Glutathione-Superflow Resin
100 ml
635608
Clontech
GST Purification Kit
5 purifications
635619
24
P R O T E I N
P U R I F I C AT I O N
Detection and purification of c-Myc and HA-tagged
proteins
c-Myc Monoclonal Antibody
HA-Tag Polyclonal Antibody
The c-myc monoclonal mouse antibody recognizes an epitope
located within residues 410–419 of the human p62-c-Myc
protein.This antibody is suitable for immunoprecipitation
of c-myc-tagged fusion proteins from cell lysates, ELISA,
immunocytochemistry of microinjected or transfected cells, and
localization of c-myc-tagged proteins.
The HA-tag antibody was raised in rabbits against a synthetic
HA peptide (YPYDVPDYA, the hemagglutinin epitope
of human influenza A virus) and purified using protein
A. This purified antibody is suitable for Western blotting,
immunoprecipitation, immunofluorescence, and ELISA
applications.
Product Information
Brand
Product
Size
Cat.No
Clontech
HA-Tag Polyclonal Antibody
100 µg
631207
Clontech
c-Myc Monoclonal Antibody
200 µg
631206
Clontech
c-Myc Monoclonal Antibody-Agarose Beads
1ml
631208
Inhibition (%)
Other purification products
80
ProteoGuard
60
Competitor P
ProteoGuard—Protease Inhibitor Cocktail
• Better protease inhibition than the leading competitor
• More flexible than tablets
• Optimized mix of five different inhibitors
40
20
0
Large-scale
Small-scale
Phosphoprotein Enrichment
Phosphoprotein
Affinity Column
• Large-Scale: Complete separation of phosphorylated and unphosphorylated proteins
using gravity-flow affinity columns
• Small-Scale: Complete kit for magnetic bead-based microscale phosphoprotein
enrichment from ANY cell or tissue samples (30 minute protocol)
• Percentage of phosphorylated proteins in final eluate: 14–17% (depends on cell type)
• Ideal for cell signaling studies (no radioactivity) or 2D-PAGE
Magnetic
bead
separation
Phosphorylated
proteins
Enriched β-casein digest
2,042 3,140
kDa kDa
0
10
15
Phosphopeptide Enrichment—Small-Scale
20
kDa
250 –
150 –
100 –
75–
50–
37–
25–
20–
25
30
Minutes
Clontech
M123
35
40
45
50
• Rapid, reliable enrichment of phosphopeptides (30 minute protocol)
• Available as magnetic beads or prepacked spin columns
• Complete kits for microscale enrichment of phosphopeptides
• Yields highly concentrated samples ideal for LC or MALDI analysis
Glycoprotein Enrichment Resin & Detection Kit
• Flexible—enrichment resin can be used with gravity-flow or FPLC columns
• Superior Performance—enrichment resin shows increased specific binding
of glycoproteins and reduced nonspecific binding
• Specific—enrichment resin enriches for low- and high-abundance serum glycoproteins
25
P R O T E I N
P U R I F I C AT I O N
Other purification products
Add
salt
Filter/
Cartridge
Thiophilic Antibody Purification Resins
Load
Wash
• Purification at neutral pH—avoid antibody aggregates
• High capacity—20–25 mg Ab/ml resin
• Broad selectivity—purify IgY, IgM, IgE, and scAb
• Yields highly stable purified antibodies
• Reusable resin
Elute in neutral buffer
(pH 7.0)
UV absorbance (215 nm)
(continued...)
β-casein digested with Mag-Trypsin
0.25
0.20
0.15
0.1
0.05
0
-0.05
Mag-Trypsin Immobilized Magnetic Trypsin
0
5
10
15
20 25 30
Minutes
35
40
45
50
• Rapid and efficient protein digestion for mass spectrometry (MS) applications
• Flexible—use the right amount of Mag-Trypsin for your application
• Eliminates trypsin contamination for downstream applications
Product Information
Brand
Product
Size
Cat.No
Clontech
ProteoGuard EDTA-Free Protease Inhibitor Cocktail
10 x 100 µl
635673
Clontech
Phosphoprotein Enrichment Kit
6 preps
635624
Clontech
Phosphoprotein Enrichment Starter Kit
1 purification
635666
Clontech
Phosphoprotein Kit—Buffer A
500 ml
635626
Clontech
TALON® PMAC Magnetic Phospho Enrichment Kit1
each
635641
Clontech
Phosphopeptide Enrichment Spin Columns
25 columns
635634
Clontech
Phosphopeptide Enrichment Buffer Kit
each
635635
Clontech
Magnetic Phosphopeptide Enrichment Kit2
each
635643
Clontech
Mag-Trypsin
5 ml
635646
Clontech
Glycoprotein Western Detection Kit
20 rxns
635648
Clontech
Glycoprotein Enrichment Resin
10 ml
635647
Clontech
Antibody Purification—Thiophillic Uniflow Resin
100 ml
635614
Clontech
Antibody Purification—Thiophillic Superflow Resin
10 ml
100 ml
635616
635617
26
Takara’s Protein Sequencing and Analysis Products
Takara offers a wide variety of Protein Fragmentation products as
well as N-terminal deblocking and sequence determination and
C-terminal sequence determination products.
Product Name
The fragmentation products are used for analysis of the primary
structure of proteins and peptides.
Application
Description
•Liberates N-terminal amino acids up to
X-Pro from proteins and peptides
Pfu Aminopeptidase I is a thermostable exo-type aminopeptidase, isolated from Pyrococcus furiosus and produced as a
recombinant protein, which liberates the N-terminal amino
acid from proteins and peptides.
N-terminal and C-terminal analysis
Pfu Aminopeptidase I
•Removal of pyroglutamic acids from
the N-terminal of proteins and peptides
Pfu Pyroglutamate
Aminopeptidase
•Deblocking of N-terminal
pyroglutamates of proteins and
peptides for sequence analysis using
Edman degradation
Pfu Methionine
Aminopeptidase
•Liberates the N-terminal methionine
residues from proteins and peptides
Pfu N-acetyl Deblocking Aminopeptidase
(Ac-DAP)
•N-Terminal deblocking
•N-terminal sequence analysis of
blocked proteins or peptides
Pfu Pyroglutamate Aminopeptidase liberates the N-terminal
pyroglutamic acid from proteins and peptides. This enzyme
may work well with some intact, non-denatured proteins
and the denaturation step may be unnecessary in these
instances.
Pfu Methionine Aminopeptidase specifically liberates only
the N-terminal methionine residue from Met-X-Y when X is
Ala, Gly, Ser, Thr, Pro or Val. This enzyme does not liberate the N-terminal Met when the N-terminus sequence is
Met-Met-Y or Met-Met-Met-Y. It is not active toward formylmethionine.
Pfu N-acetyl Deblocking Aminopeptidase (Ac-DAP) is a
unique exo-type aminopeptidase that first liberates blocking groups, such as formyl, acetyl, and myristyl, and then
releases the first and subsequent amino acids from proteins
and peptides until it reaches the first X-Pro bond.
Fragmentation of Proteins
Arginylendopeptidase
•Fragmentation of proteins and peptides required from primary structure
analysis
Arginylendopeptidase cleaves peptide bonds at the carboxyl
side of arginine residues found in pro­teins and pep­tides.
Arginylendopeptidase is also known as mouse submaxillary
pro­tease D or as mouse EGF binding protein C.
Asparaginylendopeptidase
•Fragmentation of proteins and peptides
required for primary structure analysis
Asparaginylendopeptidase specifically cleaves peptide bonds
on the carboxyl side of asparagine residues found in proteins
and peptides. Glycosylated asparagine residues are not
cleaved.
Endoproteinase Asp-N
•Fragmentation of proteins and peptides
required for primary structure analysis
Endoproteinase Asp-N is a metalloprotease that hydrolyzes
peptide bonds on the amino side of Asp and Cys oxidized to
cysteic acid. If cysteine is reduced or alkylated, the enzyme
will cleave only the amino side of Asp residues.
Pfu Protease S
•Fragmentation of proteins and peptides
required for primary structure analysis
Pfu Protease S is an endo-type serine protease with broad
recognition of native and denatured proteins. Cleavage
occurs mainly on the carboxy side of peptide bonds of
hydrophobic amino acid residues.
•Calpain protease inhibitor
Calpastatin is an endogenous protease in­hib­i­tor that acts
specifically on calpain calcium-dependent cysteine pro­tease.
It consists of four repetitive sequences of 120 to 140 amino
acid residues (domains I, II, and IV), and an N-ter­mi­nal nonhomologous sequence (L). The prod­uct consists of highly
purified recombinant human calpastatin domain I .
Protease Inhibitor
Calpastatin
Product Information
Brand
Product
Size
Cat.No
Takara
Pfu Pyroglutamate Aminopeptidase
10 mU
7334
Takara
Pfu Methionine Aminopeptidase
20 mU
7335
Takara
Pfu Aminopeptidase
0.5 mg
7336
Takara
Pfu N-acetyl Deblocking Aminopeptidase
50 µg
7340
Takara
Arginylendopeptidase
0.5 mg
7308
Takara
Asparaginylendopeptidase
0.2 mU
7319
Takara
Endoproteinase Asp-N
2 µg
7329
Takara
Pfu Protease S
500 U
7339
Takara
Calpastatin
3 mg
7316
27
EUROPE
Takara Bio Europe, SAS
2, av. du Président Kennedy
78100 St-Germain-en-Laye
France
Tel.:
UK: 0808 234 8063
Germany: 0800 182 5178
Austria: 0800 296 141
Switzerland: 0800 563 629
Europe: +33 (0)1 3904 6880
Fax:
+33 (0)1 3904 6870
Email:
[email protected]
[email protected]
[email protected]
www.takara-bio.eu
www.clontech.com
WORLDWIDE
Takara Bio, Inc. JAPAN
Clontech Laboratories, Inc. USA
A Takara Bio Company
Japan: +81.(0)77.543.724
Asia Pacific: +1.650.919.7300
United States/Canada: +1.800.662.2566